Preform produced by electrospinning, method for producing the same and use of such a preform

ABSTRACT

The invention relates to a method for producing a preform by means of an electrospinning process. The present invention also relates to the use of the present preform as a substrate for growing human or animal tissue thereon. The present invention furthermore relates to a method for growing human or animal tissue on a substrate, wherein the present preform is used as the substrate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/047,348 filed Feb. 18, 2016, which is incorporated herein byreference.

U.S. patent application Ser. No. 15/047,348 filed Feb. 18, 2016 is acontinuation of U.S. patent application Ser. No. 13/943,925 filed Jul.17, 2013, which is incorporated herein by reference.

U.S. patent application Ser. No. 13/943,925 filed Jul. 17, 2013 is acontinuation of U.S. patent application Ser. No. 11/587,641 filed Aug.22, 2007, which is incorporated by reference.

U.S. patent application Ser. No. 11/587,641 filed Aug. 22, 2007 is a 371of PCT Patent Application PCT/NL2005/000324 filed Apr. 28, 2005.

PCT Patent Application PCT/NL2005/000324 filed Apr. 28, 2005 claimspriority of NL Patent Application 1026076 filed Apr. 29, 2004.

FIELD OF THE INVENTION

The present invention relates to a method for producing a preform bymeans of an electrospinning process. The present invention also relatesto the use of a preform obtained in accordance with the present method,as well as to a method for growing human or animal tissue on asubstrate.

BACKGROUND OF THE INVENTION

US patent application No. 2002/0173213 discloses a biologicallydecomposable or absorbable fibre-like object produced byelectrospinning. Such a method can only be used for producingsubstantially flat objects, such as membranes, whereas it is preciselythree-dimensional preforms produced by electrospinning that are in greatdemand.

Consequently it is an object of the present invention to provide amethod for producing a preform by electrospinning, from which preformthree-dimensional objects can be made.

Another object of the present invention is to provide a preform that canbe used as a substrate for growing human or animal tissue thereon.

Yet another object of the present invention is to provide a substratefor an artificial implant, in particular for a heart valve or aT-connection for blood vessels.

SUMMARY OF THE INVENTION

One or more of the above objects can be accomplished by using the methodas referred to in the preamble, which is characterized by the followingsteps:

a) providing a mould made up of at least two submoulds, which submouldssubstantially exclusively comprise convex surfaces;

b) applying at least one fibre layer to the surface of at least one ofthe submoulds of step a) by electrospinning;

c) combining at least one submould of step a) and at least one submouldof step b);

d) applying at least one fibre layer to the surface of the assembly ofstep c) by electrospinning to obtain the preform.

The advantage of the present method is that it is possible to obtain apreform having any desired three-dimensional shape, using anelectrospinning process, by converting the intended three-dimensionalshape into a mould, which mould is subdivided into a number ofsubmoulds. Said submoulds have a spatial configuration such that,besides the usual flat parts, they substantially exclusively compriseconvex surfaces.

The present inventors have found that when a fibre layer is applied to atarget having a complex three-dimensional shape, viz. convex, concaveand flat parts, by means of an electrospinning process, problems occurin the forming of the fibre layer, since it appears not be possible toform a uniform fibre layer because extra fibres are formed between theconcave edges of the mould. Thus it is difficult to provide such concavesurfaces with a uniform fibre layer, which uniform fibre layer is highlydesirable in practice.

The aforesaid problem has been solved by the steps a)-d) of the presentmethod, in which the very presence of concave surfaces is avoided bysubdividing the mould into a number of submoulds, which submoulds are soconstructed that the submoulds do not have any concave shapes any morebut substantially exclusively comprise convex surfaces besides the usualflat parts.

The various submoulds of which the mould is built up are so constructedthat they can be combined to form the mould. The submoulds have one ormore surfaces that are contiguous to one or ore surfaces of the othersubmoulds, so that said submoulds fit together so as to jointly form themould.

Since the preforms that are used in practice frequently have concave aswell as convex surfaces, it has not been possible so far to produce suchcomplex three-dimensional preforms provided with a uniform fibre layerby coating the mould by means of an electrospinning process.

The present inventors have found, however, that it is possible to obtainthe desired preforms by subdividing the mould into a number ofsubmoulds, which submoulds each mainly comprise convex surfaces besidesthe usual flat parts that are already present. Subsequently, saidsubmoulds can be separately provided with fibre layers in one or moresteps, after which the submoulds provided with fibre layers can bejoined together and as a whole be provided with an additional fibrelayer so as to strengthen the whole.

The submoulds are made of a material that is suitable for use withelectrospinning, such as a metal. Also other suitable materials can beused, however.

The submoulds may be solid or partially hollow. If the submoulds arepartially hollow, they may have a closed exterior surface. The submouldsor the mould may be provided with one or more openings, in whichopenings holders can be fitted, for example, which holders can be usedfor correctly positioning the submoulds or the mould during theelectrospinning process.

The fibre layer(s) is (are) applied to the surface of thesubmould/mould, which surface is understood to be the exterior surfaceof the submould/mould. It is preferred to provide a large part of thesubmould/mould with at least one fibre layer. It is also possible,however, to provide only part of the surface of the submould/mould withat least one fibre layer. Thus it is possible, for example, not toprovide the part of the submould/mould that comprises the holder and/orthe part that is used for positioning the submould/mould during theelectrospinning process with at least one fibre layer.

In a specific embodiment of the present invention, at least two fibrelayers are applied in step b), viz, first a fibre layer V andsubsequently a fibre layer W, with the fibre layer V and the fibre layerW having mutually different biological decomposition rates. Saiddecomposition rate can be measured in accordance with standard methods,for example, which methods will not be explained in more detail herein.

Preferably, the biological decomposition of the fibre layer V takesplace more rapidly than that of the fibre layer W. In this way the outerlayer W of the common fibre layer provides the required strength, whilstthe inner fibre layer V can be substituted for natural tissue.

According to another preferred embodiment, a fibre layer N is used instep b) and a fibre layer M is used in step d), with the fibre layer Mbeing biologically decomposed more quickly than the fibre layer N. Inthis case, too, the fibre layer N functions to provide stability, whilstthe fibre layer M, which is applied as the outer layer for keeping theindividual fibre layers of the submoulds together, will decompose morerapidly.

In yet another embodiment of the present invention, more than two fibrelayers are provided, which layers can all be individually selected fromthe fibre layers V, W, N and M.

Any fibre material that can be processed by electrospinning can be usedas the material for the fibre layer. It is possible, for example, to usepolymeric materials, in particular biologically compatible polymericmaterials, as the fibre material.

Another especially preferred embodiment relates to the use of a fibrelayer comprising fibres composed of at least two components, wherein thevarious components have mutually different biological decompositionrates. The fibre consists of sequentially arranged component a andcomponent b, for example, so that a fibre exhibiting a repetitivecomposition -a-b-a-b-a-b- is obtained. When such a fibre layer is used,one of the two components will decompose after some time, so that acollection of short fibres remains, viz, the fibres of the componenthaving the slower decomposition rate. The short fibres, which are stillpresent, contribute to the mechanical strength of the newly formednatural tissue, whilst the tissue can grow, which is not possible when afibre layer that only consists of a slowly decomposing component isproduced.

An advantage of the use of a fibre layer consisting of fibres that arecomposed of two components is the fact that when a preform made of suchfibres is used for implantation into young patients, no subsequentsurgery is required for exchanging the implant for a larger implant.After all, the implant produced in accordance with the present inventioncan grow with the patient.

The present invention also relates to the use of a preform obtained withthe present method as a substrate for growing human or animal tissuethereon. The fibre layer that has been applied by electrospinning is aporous network on which cells can grow.

Subsequently, the substrate with the cells that have grown thereon canbe implanted at the intended position in the body. A number of preferredembodiments are defined in the subclaims and will be explained in moredetail hereinafter.

The present invention furthermore relates to a method for growing humanor animal tissue on a substrate, wherein the present preform is used asthe substrate. In this way the preform obtained by electrospinning canbe provided with a layer of human or animal tissue. Thus, an implantthat can be used for implantation into a human or animal body can beobtained through incubation with human or animal cells. Such incubationcan be carried out in a bioreactor, for example, in which a specificsubstrate solution is present, in which substrate solution the cells tobe grown are present. Said incubation can be carried out under suitableconditions of temperature, time, pH and the like so as to optimise thecell growth. This will be explained in more detail hereinafter.

The present invention will now be explained in more detail by means of adescription of a number of preferred embodiments, in which reference ismade to the accompanying drawings. The present invention is not limitedto such specific embodiments, however.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a shows three submoulds of a mould for an artificial,three-membrane heart valve. FIG. 1b is a sectional view of an assemblyof the submoulds of FIG. 1 a, which have been provided with a fibrelayer by electrospinning.

FIG. 2a shows an assembly of the three submoulds of FIG. 1a and acomplementary submould for obtaining a complete heart valve, whilst FIG.2b is a sectional view of the submoulds of FIG. 2a slid one intoanother. FIG. 2c is a sectional view of the entire mould for the heartvalve comprising fibre layers obtained after electrospinning.

FIGS. 3a and 3b are a top plan view and a side view, respectively, of apreform according to the present invention obtained by using the mouldof FIGS. 2a -2 c.

FIG. 4a shows a mould according to another embodiment of the presentinvention, a T-piece for connecting two or more blood vessels. FIGS. 4band 4c show two possible embodiments of a submould.

DETAILED DESCRIPTION

The present invention will now be explained in more detail withreference to the drawings, which show especially preferred embodimentsof the present invention. A preform is made, among other things, whichpreform functions as a mould for a heart valve (FIGS. 1-3). The drawingsshow a mould for a heart valve comprising three membranes; according tothe invention, however, also other types of valves comprising more orfewer membranes can be produced.

FIG. 1a shows three submoulds 1, each comprising one upper surface 2 andtwo contact surfaces 3, which submoulds are each separately providedwith a fibre layer by means of an electrospinning process. Said threesubmoulds 1 are so constructed that they substantially exclusivelycomprise convex surfaces besides the usual flat surfaces. It should beunderstood that the submould I does not have any concave surfaces, sothat said electrospinning will lead to a uniform fibre layer. Thesubmoulds 1 are configured to fit together to form the mould.

FIG. 1b is a sectional view of the three submoulds 1 of FIG. 1 a,showing the submoulds after a fibre layer 4 has been applied to each ofthe individual submoulds 1. The submoulds 1 have subsequently beencombined into an assembly 5 by placing the contact surfaces 3 intoabutment with each other. The part 6 is called the co-optation surface,which is very important in obtaining a properly functioning artificialheart valve. The fact is that such a co-optation surface ensures thatthe membranes will correctly butt together after the incubation of thepreform with human or animal cells so as to obtain the final biologicalheart valve. Since a certain degree of shrinkage of the preform mayoccur during incubation, it is important that an extra edge (theco-optation surface) is present on the membranes, so that saidco-optation surfaces 6 can prevent openings being formed between themembranes when shrinkage occurs, which openings might lead to a leakingheart valve. Such a co-optation surface is not obtained if a singlemould for a heart valve is used instead of three submoulds 1 accordingto the present invention.

FIG. 2a shows the assembly 5 of the submoulds 1 provided with a fibrelayer (not shown). The assembly 5 is held together by means of a ringconstruction 7, but it is also possible to use other, conventionalmethods, of course. Furthermore, a complementary submould 8 is shown,which can be placed on the end of the assembly 5 with a close andprecise fit.

The entire mould of the heart valve as shown in FIG. 2b consists of theassembly 5 of three submoulds 1 provided with a fibre layer, a ringconstruction 7 and the submould 8. In a next step (d) of the method, theentire mould will be provided with a fibre layer 9 by electrospinning.

FIG. 2c is a sectional view of the mould after step d), showingsubmoulds 1,8 with upper surfaces 2 and fibre layers 4,9. The figurefurthermore shows the co-optation surface 6, which forms part of thefibre layer 4, membranes 10, likewise forming part of the fibre layer 4,which membranes 10 are formed on the upper surfaces 2 of the submoulds1.

FIGS. 3a and 3b are views of the preform thus obtained after thesubmoulds 1,8 have been removed. Said submoulds can be carefully removedfrom the fibre layer (s) by one. Said removal may take place by hand,for example. In addition, part of the fibre layers 4 on the internalcontact surfaces 3 is removed, with the exception of the co-optationsurface 6, which is maintained. FIG. 3a is a top plan view and FIG. 3bis a side view of the preform after the submoulds 1,8 have been removed,showing the membranes 10, the fibre layer 4 (full line) and the fibrelayer 9 (dotted line), whilst FIG. 3b also shows the co-optation surface6.

Although the production of artificial heart valves has already beenextensively described in the literature, with US patent application No.2002/0173213 disclosing the use of artificial heart valves made of metalalloys, such heart valves have this drawback that a material that doesnot naturally occur in the body (metal alloy) is implanted into thebody, where it will permanently remain.

Consequently, the present invention has this advantage that a fibre-likepreform in the form of a heart valve can be obtained, which preformcomprises biologically decomposable components.

The obtained fibre-like preform according to the present invention canbe incubated with human or animal cells, which are able to grow in theopen fibre-like structure. To this end, the present fibre-like preformis transferred to a container in which a usual cell growth medium ispresent, to which human or animal cells are subsequently added. Then thewhole is cultured for a specific period of time under standard culturingconditions. In this way an artificial implant based on the presentfibre-like preform provided with human or animal tissue is obtained.

The implant thus obtained can be implanted into a human or animal body.There is a possibility that the preform will partially or entirelydecompose during the culturing of human or animal tissue, but there isalso a possibility of the decomposition of the preform continuing orstarting after implantation. Thus it is preferable according to theinvention to use a biologically decomposable or absorbable material forthe preform, so that the preform will have decomposed substantiallycompletely after some time and have been replaced by natural tissue, sothat a fully natural implant will be present in the body, in contrast tothe metal heart valves according to the prior art.

Another embodiment of the present invention relates to the use of thepresent preform as a substrate for connecting one or more blood vessels,as shown in FIG. 4. When one or more blood vessels are connected bysuturing, leakage frequently occurs, because this is a complex procedureand the blood vessels are so small and circular in shape that suturingis problematic. Consequently, there is a need for a T-piece that can beused for connecting two or more blood vessels.

FIG. 4a shows a mould of a T-piece 11 provided with two branches 12,13for joining two blood vessels.

FIG. 4b shows a submould 14 which, in combination with the mirror imagethereof (not shown), forms the complete mould for the T-piece. It shouldbe understood that the submould 14 does not comprise any concavesurfaces, so that the electrospinning process will provide a uniformfibre layer. The two submoulds 14 are separately provided with a fibrelayer by electrospinning, and subsequently the two submoulds providedwith the fibre layer are joined together, after which the submoulds thusjoined together are bonded to each other, for example by means of tubespreviously formed by electrospinning or by providing an extra fibrelayer by electrospinning.

An alternative to the above geometry of the submould 14 is shown in FIG.4c , in which the submould 17 consists of a flat plate 15 with the samegeometric FIG. 16 as shown in FIG. 4b present thereon. This mould willprovide a satisfactory distribution of the fibres. Once the fibre layerhas been applied to the submould 17 according to FIG. 4c byelectrospinning, the fibre layer can be removed from the plate 15 alongthe lines of the geometric FIG. 16, and subsequently the final mould asshown in FIG. 4a can be formed in the same manner as described above bybonding the fibre layers of the two submoulds together.

A preform for a T-connection 11 according to the invention obtained inthis manner can be used for growing human or animal tissue thereon andbe used for implantation in a comparable manner as described above withregard to heart valves.

Although the present invention has been explained on the basis of twopreferred embodiments, it is also possible to use the present inventionfor producing other preforms to be used in the production of implantsfor other parts of the body, such as other valves in the heart or bloodvessels, or parts of joints, for example a kneecap, and the like.

What is claimed is:
 1. A method for implanting a heart valve or bloodvessel preform, comprising: providing an electrospun, porous,biodegradable preform having membranes produced by electrospinning atleast two fibre layers over submoulds, wherein at least one of thesubmoulds is incorporated into the preform, the preform having an openfiber-like structure; and implanting the preform in a human or animalbody, wherein the preform decomposes substantially completely and isreplaced by natural tissue, and wherein the preform forms at least partof a blood vessel or a heart valve.
 2. The method as in claim 1, whereinthe preform is incubated with human or animal cells prior to incubationto allow cells to grow in the open fiber-like structure to providetissue over the preform.
 3. The method as in claim 1, wherein thepreform at least partially degrades prior to implantation.
 4. The methodas in claim 1, wherein the preform entirely degrades prior toimplantation.
 5. The method as in claim 1, wherein the two fibre layershave different decomposition rates.
 6. The method as in claim 1, whereinthe at least one of the submoulds is incorporated into the preform as asupport structure.
 7. A heart valve or blood vessel preform, comprising:an electrospun, porous, biodegradable preform having membranes producedby electrospinning at least two fibre layers over submoulds, the preformhaving an open fibre-like structure, wherein at least one of thesubmoulds is incorporated into the preform, and the preform beingconfigured to decompose substantially completely and be replaced bynatural tissue after implantation into a human or animal body, andwherein the preform forms at least part of a blood vessel or a heartvalve.
 8. The preform as in claim 7, wherein the preform is configuredto allow human or animal tissue to grow in the open fibre-like structureas a result of incubation with human or animal cells prior toimplantation.
 9. The preform as in claim 7, wherein the preform isconfigured to at least partially degrade prior to implantation.
 10. Thepreform as in claim 7, wherein the preform is configured to entirelydegrade prior to implantation.
 11. The preform as in claim 7, whereinthe two fibre layers have different decomposition rates.
 12. The preformas in claim 7, wherein the at least one of the submoulds is incorporatedinto the preform as a support structure.